Phosphorylated derivatives of phosphatidylinositol, collectively known as phosphoinositides (PIs), play a key role in the membrane recruitment and activation of cytosolic proteins involved in cell signaling and vesicle trafficking. The majority of PI-responsive proteins contain one or more modular lipid binding domains that specifically recognize PIs. Some of these PI-binding domains not only bind PIs but also induce the deformation of PI-containing membranes. Despite the importance of PI-mediated membrane targeting in health and disease, fundamental understanding of complex mechanisms by which PIs differentially and specifically mediate diverse cellular functions through interactions with these effector domains is still largely lacking. The primary objective of this proposed research is to elucidate the mechanisms by which various PIs mediate cellular processes through interactions with three major types pf effector domains, PX, ENTH, and BAR domains. During the past grating period, we discovered that PIs specifically induce the membrane penetration of many effector domains, thereby modulating the cellular functions and regulation of proteins harboring the domains. We have also developed several new methodologies that will allow for both large-scale and in-depth mechanistic studies of PI-binding and membrane-deforming proteins. With a new paradigm and new methodologies, we will pursue three specific aims in the next project period. First, based on the hypothesis that mammalian PX domains mediate diverse cellular processes through their divergent PI specificities and membrane binding properties, we will determine membrane binding properties of PX domains both comprehensively and systematically to elucidate the complex mechanisms of diverse PX domain-mediated cellular function and regulation. Second, we will investigate the mechanisms by which various PIs induce the membrane penetration, protein self-association, and membrane deformation by ENTH domains. Finally, we will determine how differently several BAR domains and ENTH domains penetrate and deform membranes to understand the basis of complex and highly orchestrated actions of a large number of cytosolic proteins during cellular membrane remodeling. A long-term objective of this program is to apply the principles learned from these studies to the development of new classes of therapeutic agents that can specifically modulate the membrane targeting and activation of PI-binding proteins essential for cell signaling and membrane remodeling. Principal methodologies to be used in these proposed studies are a wide range of biophysical, computational, and structural techniques as well as cell imaging by fluorescence microscopy. PUBLIC HEALTH RELEVANCE: Numerous human diseases, including cancer, diabetes, and inflammatory diseases, are known to be linked to defects in phosphoinositide-mediated cell signaling. Consequently, phosphoinositide signaling pathways are major targets for drug development but the complexity of these pathways has hampered the drug discovery effort. Therefore, understanding the mechanisms underlying complex phosphoinositide signaling pathways will greatly aid in developing new classes of therapeutic agents that can treat diseases caused by dysfunctional phosphoinositide signaling pathways.